CN111366563B - Digital plasma immunoadsorption kit and manufacturing and testing method thereof - Google Patents

Digital plasma immunoadsorption kit and manufacturing and testing method thereof Download PDF

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CN111366563B
CN111366563B CN202010173864.9A CN202010173864A CN111366563B CN 111366563 B CN111366563 B CN 111366563B CN 202010173864 A CN202010173864 A CN 202010173864A CN 111366563 B CN111366563 B CN 111366563B
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CN111366563A (en
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刘钢
张维
党棠
胡文君
许浩
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Liangzhun Shanghai Medical Devices Co ltd
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Abstract

The invention provides a digital plasma immunoadsorption kit and a manufacturing and testing method thereof. The digital plasma immunoadsorption kit comprises: the kit comprises a box body, a nano plasma sensor chip and a reagent, wherein the nano plasma sensor chip and the reagent are arranged in the box body; the surface of the nano plasma sensor chip is provided with a nano cup array, and each nano cup in the nano cup array comprises a nano column, and a titanium layer and a gold layer which are sequentially formed on the surface of the nano column.

Description

Digital plasma immunoadsorption kit and manufacturing and testing method thereof
Technical Field
The invention relates to a digital plasma immunoadsorption kit for ultrahigh-sensitivity protein affinity determination and dynamic interaction combined imaging.
Background
Surface Plasmon Resonance (SPR) biosensors have high sensitivity to changes in local Refractive Index (RI) due to the evanescent field generated by them, and have become important tools for studying the kinetics of biomolecular interactions. The proposal of Surface Plasmon Resonance (SPR) imaging is to combine SPR technology with an imaging system closely, and become a technology with the characteristics of no mark, high flux, in-situ and real-time detection of molecular interaction and the like. Can be used to measure specificity, affinity and kinetic parameters in macromolecular binding processes, such as protein-protein, protein-DNA, receptor drug and cell/virus-protein binding. Are increasingly used in early diagnosis of diseases, drug screening and food quality control.
The concept of digital PCR (polymerase chain reaction) has attracted continuous attention since its first introduction in 1999, and dPCR provides a more sensitive and reproducible clinical approach than real-time quantitative PCR (qpcr). Digital pcr (dpcr) is an increasingly widely used technique with many advantages in the detection and quantification of nucleic acids. In recent years, with the development of microfluidic and emulsion chemistry, the simplified and automated process has made dPCR more practical and increasingly used in clinical assays, including oncology and infectious diseases as well as fetal gene screening and predicting transplant rejection.
However, the conventional detection device has problems of complicated operation and expensive equipment, and is not favorable for popularization of detection means.
Disclosure of Invention
The invention aims to solve the technical problem of the prior art, and provides a digital plasma immunoadsorption kit for ultrahigh-sensitivity protein affinity determination and dynamic interaction combined imaging, which avoids the use of expensive spectrum detection instruments, realizes bright-field imaging only by using a common white light source and photographing equipment, and has no complicated and lengthy signal amplification step.
According to the present invention, there is provided a digital plasma immunoadsorption kit comprising: the kit comprises a box body, a nano plasma sensor chip and a reagent, wherein the nano plasma sensor chip and the reagent are arranged in the box body; the surface of the nano plasma sensor chip is provided with a nano cup array, and each nano cup in the nano cup array comprises a nano column, and a titanium layer and a gold layer which are sequentially formed on the surface of the nano column.
Preferably, the reagent comprises: sucrose, hexylsilane, ethanol, 11-mercaptodecanoic acid, 1-ethyl-3- (dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, bovine serum albumin, ethanolamine, purified CRP and phosphate buffer, monoclonal anti-CRP capture and detection antibodies.
According to the present invention, there is also provided a method of manufacturing the above-mentioned digital plasma immunoadsorption kit, comprising manufacturing a nanoplasmon sensor chip by the following method: manufacturing an original mold by a tapered nano-pillar array manufactured on a silicon oxide wafer through laser interference lithography; silanization of the original mold to obtain hydrophobicity before replication of the original mold; then, coating an optical adhesive on a clean original mold, placing a PET sheet on the top of the mold, and stripping a PET sheet with a nanopore array on the surface from the mold after ultraviolet irradiation; thereafter, a titanium layer and a gold layer were deposited on the PET surface having the nanopore array using an electron beam evaporator to fabricate a nanoplasmon sensor chip.
According to the invention, the invention also provides a test method adopting the digital plasma immunoadsorption kit, which comprises the following steps: cleaning the surface of a nano plasma sensor chip, collecting a transmission spectrum and a picture in deionized water, and recording a collection area; adding 11-mercapto decanoic acid solution to the surface of the core nano plasma sensor sheet in ethanol, incubating to remove the 11-mercapto decanoic acid solution, and then cleaning the chip by using ethanol and deionized water; then collecting the transmission spectrum and the picture of the recording area in deionized water; the chip was incubated in a 1:1 mixture of EDC and NHS for a predetermined time, then immediately incubated with a monoclonal anti-CRP capture antibody in PBS at a predetermined temperature; cleaning the surface of the chip, and then collecting the transmission spectrum and the picture of the recording area in deionized water; incubating the chips in the BSA blocking solution and the ethanolamine solution for a preset time; washing the chip with deionized water, and then collecting the transmission spectrum and the picture of the recording area in the deionized water; incubate the chips in PBS in different concentrations of CRP; collecting a transmission spectrum and a picture of a recording area in deionized water after washing with PBS and the deionized water; finally, adding a monoclonal anti-CRP detection antibody to the surface of the chip in PBS, and collecting dynamic pictures of the recording area changing along with time; the chip was rinsed with PBS and deionized water, and transmission spectra and pictures of the recorded area were collected in deionized water.
According to the invention, the invention also provides a CRP protein concentration sandwich test method adopting the digital plasma immunoadsorption kit, which comprises the following steps: firstly, forming SAM (self-assembled monolayers) on the surface of the chip in 11-mercaptodecanoic acid solution, activating carboxyl with EDC and NHS, and binding CRP capture antibody to the surface of the chip; blocking non-specific sites with BSA and ethanolamine solution and covering the remaining NHS; then adding the CRP protein solution to the surface of the chip, so that the CRP protein free in the solution is captured by the CRP antibody fixed on the surface of the chip and remains on the surface of the chip; (ii) allowing the concentration of CRP in the solution to be below a predetermined concentration, wherein on average each nanopore comprises one to a plurality of molecules of CRP protein; the CRP protein is randomly dispersed to all positions on the surface of the chip and captured, so that the CRP protein is distributed on the surface of the chip unevenly; finally, a predetermined amount of CRP detection antibody is added to the surface of the chip to be combined with the captured CRP protein, so that the combination of the CRP detection antibody on the surface of the chip changes the RI of the environment in the area where the CRP detection antibody is located to enable the plasma resonance peak to be red-shifted, and thus the area on the surface of the chip is divided into two parts, the area containing the CRP protein changes the original SPR signal after being combined with the CRP detection antibody, and the area without the CRP protein can not be combined with the CRP detection antibody to keep the original SPR signal.
Preferably, a poisson distribution equation is introduced:
Figure GDA0003402664790000031
wherein λ is a parameter associated with the concentration c of the CRP protein, and is k1*c，k1Is a coefficient; the probability P represents the ratio of the nanopore region where the SPR signal changes to the detected nanopore region; the approximation of the ratio of the nanopore region is represented by the change in the number of pixel points, and the rate of change in the number of pixel points where the SPR signal changes is represented as p, i.e., p
Figure GDA0003402664790000032
Ncp represents the number of changed pixels, Ntot represents the total number of pixels; adding a correction factor k2To establish the corresponding relationship between the region where the SPR signal changes and the change of the number of pixel points, there is a formula of the corresponding relationship:
p＝k2(1-|k1×c)；
taking logarithms at two sides to obtain a concentration formula:
c＝k1×In(1-k2×p)。
preferably, the corresponding relation formula is used for fitting the function relation of the change rate p of the number of the pixel points with the CRP concentration, and the fitting result when the CRP concentration is lower than 50ng/ml is taken to determine the coefficient k1、k2To further express the concentration formula as C (CRP) 0.10804 × In (1-4.90938 × p) to calculate the CRP protein concentration.
Preferably, the test method is for the detection of the inflammation marker CRP protein.
Preferably, a halogen lamp is used for illumination, and the image in transmission mode is exposed on the CCD camera through a condenser lens and a magnifying objective lens.
The invention introduces the digital PCR concept into the SPR imaging technology for the first time, and realizes the quantitative detection of the CRP protein concentration by the digital SPR image technology by combining the classical Poisson statistical algorithm. Meanwhile, the binding kinetics of the CRP and the anti-CRP antibody were determined by collecting dynamic SPR images of the protein binding process. Different from the traditional method for calculating the mean value of analog signals of an SPR image, the digital signal image is detected by directly counting the changed sensitive pixel points, and the conversion of the SPR image from an initial image to the analog signal image and then to the digital signal image is realized by an automatic image processing mode, so that the improvement of 10 times of sensitivity is realized. The method is used for quantitatively detecting the concentration of human C-reactive protein (CRP, 100kDa) in a buffer solution, and the CRP is a biomarker widely used for clinical diagnosis and treatment of acute inflammatory diseases. Here, it is demonstrated that the limit of detection (LOD) is as low as 1 ng/ml. This result was at least 3 orders of magnitude lower than plasma CRP levels. The research of the invention provides a new idea and a new method for detecting the protein concentration by Surface Plasmon Resonance (SPR) imaging. Meanwhile, the method avoids the use of expensive spectrum detection instruments, realizes bright field imaging only by using a common white light source and photographing equipment, does not have complicated and lengthy signal amplification steps, and is expected to develop a portable biosensor for early detection of diseases.
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A more complete understanding of the present invention, and the attendant advantages and features thereof, will be more readily understood by reference to the following detailed description when considered in conjunction with the accompanying drawings wherein:
FIG. 1 schematically shows a schematic diagram of a digital plasma immunoadsorption kit for ultra-high sensitivity protein affinity assay and dynamic interaction binding imaging according to a preferred embodiment of the present invention.
FIG. 2a schematically illustrates a digitized image protein detection schematic of a digitized plasma immunoadsorption kit for ultra-high sensitivity protein affinity assay and dynamic interaction binding imaging according to a preferred embodiment of the present invention.
FIG. 2b schematically shows that the diluted CRP proteins are randomly dispersed throughout the chip surface and bound to capture antibodies immobilized on the chip surface, thereby attaching to the chip surface; the chip surface will now be divided into a region containing the CRP protein and a region not containing the CRP protein.
FIG. 2c schematically shows that excess detection antibody is dripped onto the chip surface and bound to the captured CRP protein for signal amplification, and the attachment of the detection antibody causes a large red-shift of the plasmon resonance peak.
Fig. 2d schematically shows the statistical counting of pixel points for the region containing CRP protein and the region not containing CRP protein using image processing means.
Figure 2e schematically shows the derivation of a digital SPR algorithm in combination with a classical digital PCR poisson distribution algorithm.
FIG. 2f schematically shows the determination of the correlation coefficient by exponential fitting of 1-50ngCRP proteins using the deductive formula.
FIG. 3 schematically shows the SPR image processing analysis flow of the digital plasma immunoadsorption kit test method for ultra-high sensitivity protein affinity assay and dynamic interaction binding imaging according to a preferred embodiment of the present invention.
FIGS. 4a, 4b and 4c show the variation of the mean value of the analog signal and the count value of the digital signal with time in the dynamic image detection of CRP protein at 1ug/ml, 100ng/ml and 10ng/ml respectively; fig. 4d shows a bar graph showing the change in the mean value of the simulated signal and the count value of the digital signal (LOD 1ng/ml) for different concentrations of CRP samples after reaction with the detection antibody; fig. 4e shows plots of the analogue signal mean and digital signal count values as a function of CRP concentration plotted on a logarithmic scale, with the slope of the digital signal count value (fitting coefficient 0.985) being 10 times the slope of the analogue signal mean value (fitting coefficient 0.977); FIG. 4f shows a graph of the digital signal counts as a function of time for the dynamic image detection of CRP proteins at different concentrations.
It is to be noted, however, that the appended drawings illustrate rather than limit the invention. It is noted that the drawings representing structures may not be drawn to scale. Also, in the drawings, the same or similar elements are denoted by the same or similar reference numerals.
Detailed Description
A nanopore biosensor-based digital plasma immunoadsorption assay is presented herein. The method has high sensitivity to low-concentration protein detection. Similar to digital PCR detection, the digital SPR image analysis method in combination with the poisson distribution algorithm can be used for quantitative detection of low-concentration proteins. The invention proves that the protein binding information in the Surface Plasma Resonance (SPR) image can be effectively obtained by using the method, and compared with a method for calculating the mean value of an analog signal image, the digital signal counting method can improve the sensitivity by more than 10 times. Meanwhile, the technology of the invention avoids complex spectrum detection, and the method is applied to the detection of inflammation marker CRP protein, and the LOD is 1 ng/ml. The present invention measures protein binding kinetics by ultrasensitive dynamic imaging of the interaction of CRP protein with anti-CRP antibodies. The research of the invention provides a new idea and a new method for detecting the protein concentration by Surface Plasmon Resonance (SPR) imaging. Meanwhile, the method avoids the use of expensive spectrum detection instruments, realizes bright field imaging only by using a common white light source and photographing equipment, does not have complicated and lengthy signal amplification steps, and is expected to develop a portable biosensor for early detection of diseases.
In order that the present disclosure may be more clearly and readily understood, reference will now be made in detail to the present disclosure as illustrated in the accompanying drawings.
< composition of kit >
FIG. 1 schematically shows a schematic diagram of a digital plasma immunoadsorption kit for ultra-high sensitivity protein affinity assay and dynamic interaction binding imaging according to a preferred embodiment of the present invention.
As shown in FIG. 1, the digital plasma immunoadsorption kit for ultra-high sensitivity protein affinity assay and dynamic interaction binding imaging according to the preferred embodiment of the present invention comprises: the kit comprises a box body 11, a nano plasma sensor chip 12 and a reagent 13, wherein the nano plasma sensor chip 12 and the reagent are arranged in the box body; the nano-cup array is formed on the surface of the nano-plasma sensor chip 12, and each nano-cup in the nano-cup array comprises a nano-pillar, and a titanium layer and a gold layer which are sequentially formed on the surface of the nano-pillar.
In a preferred example, the reagent 13 comprises: sucrose, hexylsilane, ethanol, 11-mercaptodecanoic acid (MUA), 1-ethyl-3- (dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), Bovine Serum Albumin (BSA), ethanolamine, purified CRP and Phosphate Buffered Saline (PBS) buffer, monoclonal anti-CRP capture and detection antibodies.
< example of production of nanoplasmon sensor chip >
In a preferred embodiment, the nanoplasmon sensor chip can be fabricated using a replica molding process. The original mold was fabricated by laser interference lithography of an array of tapered nanopillars fabricated on a silicon oxide wafer. Prior to replication of the original mold, the original mold was silanized to obtain hydrophobicity. Then, an optical adhesive (e.g., Norland optical adhesive, NOA-61) is coated uniformly and bubble-free on the clean original mold, and then a PET (polyethylene terephthalate) sheet is placed on top of the mold. Ultraviolet light for, e.g., 90 seconds (e.g., 105 mW/cm)2) After irradiation, the PET sheet having the nanopore array on the surface was carefully peeled off from the mold. Finally, 9nm titanium and 110nm gold were deposited on the PET surface with nanopore array using an electron beam evaporator to make a nanoplasmon sensor chip. The resulting nanocup array has a period of 350nm, a diameter of 180nm and a height of 500 nm.
< preferred embodiment of test method >
The surface of the nanoplasmon sensor chip is first cleaned with, for example, 70% ethanol solution and deionized water, and then transmission spectra and pictures are collected in deionized water, and the collection area is recorded. Adding 1mM 11-mercapto decanoic acid solution (in ethanol) to the surface of the core nano-plasma sensor chip, incubating at room temperature for 24 hours, for example, removing the 11-mercapto decanoic acid solution, and then washing the chip with 70% ethanol and deionized water (for example, twice). The transmission spectrum and pictures of the recorded area were then collected in deionized water. The chip was incubated in a 1:1 mixture of EDC (e.g., 400mM) and NHS (e.g., 100mM) for 30 minutes at room temperature, then immediately incubated with 50. mu.g/ml of monoclonal anti-CRP capture antibody (in PBS) for 12h at 4 ℃. The surface of the chip is cleaned by PBS and deionized water, and then the transmission spectrum and pictures of the recording area are collected in the deionized water. The chips were incubated for 30 minutes in 60. mu.g/ml BSA blocking solution followed by 10% ethanolamine solution. The chip was rinsed twice with deionized water and the transmission spectra and pictures of the recorded areas were collected in deionized water. The chips were incubated in different concentrations of CRP (in PBS) for 2 hours. Transmission spectra and pictures of recorded areas were collected in deionized water after rinsing with PBS and deionized water. Finally, a monoclonal anti-CRP detection antibody (in PBS) at 50. mu.g/ml was added to the chip surface and a dynamic picture of the recorded area was taken over time. After 2 hours the chip was rinsed with PBS and deionized water, and transmission spectra and pictures of the recorded area were collected in deionized water.
Note that, as those skilled in the art will understand, the specific solution concentration content and the specific time data in the above flow chart can be adjusted appropriately, and the above specific values are only preferred examples of the values.
< example: digital SPR signal detection CRP protein content >
FIG. 2a schematically shows an image protein detection schematic of a digital plasma immunoadsorption kit for ultra-high sensitivity protein affinity assay and dynamic interaction coupled imaging according to a preferred embodiment of the present invention. Wherein the pentagonal-shaped icon represents CRP and "Y" represents CRP capture antibody.
Specifically, for example, as shown in FIG. 2a, the experiment of the present invention can be performed on a fluorescence inverted microscope, illuminated with a conventional 100W halogen lamp, and the transmission mode image recorded after exposure to 2448 × 1920 pixel true color CCD camera for 25ms (FIG. 2 a). Using a 0.3NA condenser and a 0.3NA, 10 times magnification objective, coverage of 844.6X 662.4um can be captured2An image of the region. From this it can be calculated that the size of the chip surface area covered by each pixel is about 345 x 345nm2This is approximately 350nm of the period of the nanopore array.
The concentration of the CRP protein is detected by adopting a sandwich method. Briefly, a self-assembled monolayer, SAM for short, was first formed on the chip surface in 11-mercaptodecanoic acid solution, and after carboxyl groups were activated with EDC and NHS, the CRP capture antibody was bound to the chip surface. Non-specific sites were blocked with BSA and ethanolamine solution and the remaining NHS was covered. The diluted CRP protein solution was then added to the chip surface, and the free CRP protein in the solution was captured by the CRP antibody immobilized on the chip surface and left on the chip surface (fig. 2 b). When the concentration of CRP in the solution is low, on average, one to multiple molecules of CRP protein per nanopore are contained. The CRP protein was randomly dispersed throughout the chip surface and captured, resulting in non-uniformity of CRP protein distribution on the chip surface (fig. 3). Finally, excess CRP detection antibody was added to the chip surface to bind to the captured CRP protein (fig. 4 c). Binding of the CRP detection antibody to the chip surface changes the RI of the environment at the region to which it is bound, causing a red shift of the plasmon resonance peak, so that the chip surface region will be divided into two portions, the region containing the CRP protein changes the original SPR signal upon binding to the CRP detection antibody, and the region not containing the CRP protein cannot bind to the CRP detection antibody to maintain the original SPR signal (fig. 4 c).
Digital PCR (dPCR) is a novel technology for absolutely quantifying and amplifying nucleic acid molecules, and because two or more CRP protein molecules can be combined in each nanopore, the invention introduces a Poisson distribution formula (figure 2e) to detect the content of low-concentration CRP protein in combination with the concept of digital PCR.
Figure GDA0003402664790000081
In this case, λ is related to the CRP protein concentration c, and is k1C. When k is 0 (no CRP protein molecule), the above formula can be simplified to:
Figure GDA0003402664790000084
the probability P can be considered here as the ratio of the nanopore region where the SPR signal changes to the nanopore region detected. The ratio of the nanopore region can be approximately represented by the change in the number of pixel points, and the present invention represents the rate of change in the number of pixel points where the SPR signal changes as p, i.e., p
Figure GDA0003402664790000082
Ncp represents the number of pixels that change, and Ntot represents the total number of pixels. Meanwhile, due to the existence of diffraction limit, the area where the SPR signal changes is not in one-to-one correspondence with the change of the number of pixel points, and a correction coefficient k needs to be added2To establish the corresponding relationship, there are:
Figure GDA0003402664790000083
expressing this as a function of p and c yields:
p＝k2(1-|k1×c) (2)
the two-sided log-taking can be further varied into a general formula:
c＝k1×In(1-k2×p) (3)
the rate of change p of the number of pixel points as a function of CRP concentration is shown in fig. 2 f. By fitting according to equation (2), it was found that when the concentration of CRP is below 50ng/ml, the fitting result is better, 0.983. From which the coefficient k can be determined1、k2Thus, the formula (3) can be expressed as c (crp) 0.10804 × In (1-4.90938 × p).
< simulation setup example >
Example a commercially available three-dimensional finite difference time domain (3D-FDTD) method was used to study the relevant changes in the far-field transmission spectra of the nanocup chips. Example a nanocup was constructed on a substrate having a thickness of 1000 nm. The period of the nanocup array was 350nm, and the top diameter, bottom diameter and depth of the nanocups were 180nm, 160nm and 500nm, respectively. A 9nm Ti layer and a 110nm Au layer (optical parameters Johnson and Christy) were placed in sequence over the cup. The light source is placed above the nanocup structure and propagates to the substrate in the normal incidence direction. The simulated total area was 350nm x 350nm x 2000 nm. Periodic boundary conditions are applied along the x and y directions and perfectly matched layers are applied along the z axis to eliminate any interference at the boundary.
< example of optical setup >
Microscopic measurements were performed using an Olympus IX73 vertical fluorescence microscope operating in transmission mode. A100W halogen lamp was used as the light source, and the optical path included a piece of ground glass, a condenser lens with NA of 0.3, and an objective lens with NA of 0.3 at 10 times magnification. The RGB true color pictures were collected by a Charge Coupled Device (CCD) camera controlled by cellSens software with an exposure time set to 25ms and a digital gain of 1. The transmission spectrum is acquired using a spectrometer (iHR320 HORIBA). The transmission spectra from the samples were normalized with the light source spectrum to obtain the final transmission data. The collection of spectral data was controlled by a custom LabVIEW program.
< example of image processing >
All the acquired RGB images are preprocessed by ImageJ software, rigid registration is carried out on the images by using a Register Virtual Stack function built in the software, and the registered images are cut into images with the size of 1000 x 1000 pixels to serve as initial images.
Subsequent image processing and analysis were performed in MATLAB software. The analog signal image is obtained by calculating the ratio of the R channel intensity value to the sum of the three channel intensity values at each pixel point of the initial image, and by this simple conversion, the variation in the overall intensity of the image caused by the fluctuation of the light source and the sample is eliminated to some extent. A more reliable signal with plasma characteristics is thus obtained. The average analog signal value is obtained by calculating the change rate of the overall average value of the analog signal image:
Vmas＝(Mean2-Mean1)/(Mean1)
mean1 and Mean2 are the overall means of the simulated signal images taken before and after addition of the detection antibody, respectively.
The digital signal image is obtained by binarizing an analog signal image by setting a threshold value. Setting a proper threshold, setting the value of the pixel point which is larger than the threshold as 1, setting the value of the pixel point which is smaller than the threshold as 0, and dividing the image into an area with the pixel value of 1 and an area with the value of 0. The digital signal count value is obtained by calculating the rate of change in the number of pixel points in the digital signal image whose pixel value is 1:
Vdsc＝(Count2-Count1)/Count1
count1 and Count2 are the number of pixel points with pixel value 1 in the digital signal image obtained before and after the detection antibody is added.
A built-in MATLAB color map (authn) is used for the visualization of the analog signal image and the digital signal image.
< SPR image optical Properties and processing >
In order to research the optical characteristics of the chip in the solution, the invention prepares 0-40% sucrose solution, which respectively correspond to RI values of 1.33-1.4. The invention collects the transmission spectrum and the microscopic picture of the same chip under sucrose solutions with different concentrations (the specific configuration is shown in the specific example). As RI increases, the transmission spectrum is red-shifted due to the red-shifted nature of the nanopore plasma. The transmission spectrum of the sensor chip under different environments RI is simulated by using three-dimensional Finite Difference Time Domain (FDTD) software, and specific simulation details can be found in the supporting information. The characteristic of the nanopore plasma is confirmed through the simulation result and the experimental result.
The conventional detection method based on the spectral peak displacement needs to rely on expensive precise instruments, and the invention can replace a spectrometer with a taken microscopic picture for detection. In the absence of an internal reference, fluctuations in the light source and the sample tend to cause variations in the intensity of the transmitted light, resulting in inaccuracies in the detection results. Therefore, the invention establishes a close relation between SPR resonance peak shift and the change of the color of the microscopic picture by normalizing the intensity value of RGB three channels at each pixel point, and obtains a stable and reliable signal image (see a specific configuration in a specific example). After normalization, the average value of the intensity values of the RGB three channels changes along with the RI, and the result shows that the normalized intensity value has a good linear relation with the RI. As the ambient RI increases, the R-channel normalized intensity value increases and the G-channel normalized intensity value decreases. Since the blue light component is very small in transmitted light, the normalized intensity value of the B channel is small, about 1%, and negligible. The normalized intensity values for both the R and G channels can be used to detect changes in RI. In the example, the R channel with a large normalized intensity value is selected to represent the SPR signal, and an analog signal image is constructed for detection. The average analog signal value is obtained by calculating the change rate of the average value of the whole analog signal image, and the calculation method is shown in a specific example.
For example, the micrograph taken was preprocessed by imageJ (see specific examples) to obtain an initial picture of 1000 × 1000 pixels, which included the chip surface area of 345 × 345um 2. Image preprocessing is necessary, which improves the accuracy of detection and the operation speed of subsequent image processing.
After the initial picture is imported into matlab software, the method is used for calculating the R/RGB value at each pixel point to serve as the SPR signal value at each pixel point, and the converted SPR signal can accurately reflect the analog signal image of the SPR signal. The initial picture and the analog signal picture have weak changes in CRP detection, and are difficult to distinguish with naked eyes. The present invention further processes the analog signal picture using a binarization method to convert it to a digital signal picture). The invention selects a proper threshold value, sets the value of the pixel point higher than the threshold value as 1, and sets the value of the pixel point lower than the threshold value as 0. The threshold is dynamically selected, and in consideration of the difference between chips used in the experiment, the same method is adopted instead of the same threshold for pictures taken under different chips. In order to ensure the stability of the extracted signal, the invention uses the median value of the analog signal picture taken before adding the detection antibody as a threshold value, and the picture taken after adding the detection antibody uses the same threshold value, after the detection antibody is combined with the CRP protein, the value of the environmental RI changes, and the plasma resonance peak is red-shifted. The R/RGB values of some pixel points in the image are increased, the pixel points which are originally lower than the threshold value cross the threshold value to become pixel points with the value of 1, the number of the pixel points with the value of 1 is increased, the CRP concentration is higher, the CRP-containing area on the surface of the chip is larger, the number of the pixel points in transition is larger, and therefore the relation between the CRP protein concentration change and the pixel point number change is constructed.
< method comparison and kinetic quantitation >
The invention uses the analog signal image and the digital signal image converted from the initial image in the detection of CRP protein concentration. FIG. 4d shows the results of measuring the concentration of CRP protein by both the analog signal and the digital signal. The LOD of the sample is 10ng/ml by using a method of simulating a signal image. The LOD of the detection is 1ng/ml by using a digital signal image method, and the detection limit is improved by 10 times. This may be due to the digitization method removing some of the background signal interference. FIG. 4e shows the curves of the average analog signal value (Vmas) and the digital signal count value (Vdsp) as a function of the CRP concentration (from 1ng/ml to 500ng/ml) on a logarithmic scale, the results corresponding to a linear relationship. The curve of the mean analog signal value (Vmas) as a function of the CRP concentration, the fitting coefficient (R) can be seen2) It was 0.977 and the slope was 0.28. Curve of digital signal count value (Vdds) with CRP concentration, fitting coefficient (R)2) It was 0.985, and the slope was 2.93. Compared with the change rate of an analog signal image, the change rate and the sensitivity of a digital signal image are improved by more than 10 times.
Fig. 4a, 4b, 4c show the average simulated signal value (Vmas) and the digital signal count value (Vdsc) over time after addition of the CRP detection antibody. It can be seen that the rate of change of the digital signal is much greater than the rate of change of the analog signal when the CRP protein is detected at the same concentration. And the reaction rate was significantly faster at 1ug/ml CRP (FIG. 4a) than at 100ng/ml CRP (FIG. 4b), and 10ng/ml CRP (FIG. 4c) during the first 30min of the reaction. SPR systems have been investigated to provide intermolecular interaction rates and to determine kinetic constants.
Figure GDA0003402664790000121
The dynamic digital signal image is applied to CRP protein binding kinetic analysis. Values of kinetic constants were determined (fig. 4f) and the fitting function was y ═ a (1-e)bx). It can be seen from the figure that the CRP fit curves of 25. mu.g/ml and 50. mu.g/ml almost overlap, indicating that saturation is reached when 50. mu.g/m CRP is added. The biomolecular interaction equation between CRP protein and Detection Antibody (DA) can be expressed as:
here, the
Figure GDA0003402664790000122
The value of (D) can be determined by measuring the digital signal count value at which the chip reaches saturation under 50ug/ml CRP. Better (R) by calculating the fitting coefficient2> 0.94) and away from saturation (< 90%
Figure GDA0003402664790000123
) The fitted curve of 0.5ug/ml and 1ug/ml CRP is obtained to obtain kaAnd k isdCalculated values are 3.994 × 10 respectively6(±0.36×106) And 2.15X 102(±0.21×102). Dissociation equilibrium constant KDEquation KD＝kd/kaThis gave a calculated value of 5.38 × (±. 0.31 ×) M. KDThe calculated value of (a) is close to the result measured by the nano particle amplification SPR imaging sensor.
The invention introduces the digital PCR concept into the SPR imaging technology for the first time, and realizes the quantitative detection of the CRP protein concentration by the digital SPR image technology by combining the classical Poisson statistical algorithm. Meanwhile, the binding kinetics of the CRP and the anti-CRP antibody were determined by collecting dynamic SPR images of the protein binding process. Different from the traditional method for calculating the mean value of analog signals of an SPR image, the digital signal image is detected by directly counting the changed sensitive pixel points, and the conversion of the SPR image from an initial image to the analog signal image and then to the digital signal image is realized by an automatic image processing mode, so that the improvement of 10 times of sensitivity is realized. The method is used for quantitatively detecting the concentration of human C-reactive protein (CRP, 100kDa) in a buffer solution, and the CRP is a biomarker widely used for clinical diagnosis and treatment of acute inflammatory diseases. Here, it is demonstrated that the limit of detection (LOD) is as low as 1 ng/ml. This result was at least 3 orders of magnitude lower than plasma CRP levels. The research of the invention provides a new idea and a new method for detecting the protein concentration by Surface Plasmon Resonance (SPR) imaging. Meanwhile, the method avoids the use of expensive spectrum detection instruments, realizes bright field imaging only by using a common white light source and photographing equipment, does not have complicated and lengthy signal amplification steps, and is expected to develop a portable biosensor for early detection of diseases.
It should be noted that the terms "first", "second", "third", and the like in the description are used for distinguishing various components, elements, steps, and the like in the description, and are not used for indicating a logical relationship or a sequential relationship between the various components, elements, steps, and the like, unless otherwise specified.
It is to be understood that while the present invention has been described in conjunction with the preferred embodiments thereof, it is not intended to limit the invention to those embodiments. It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.

Claims (5)

1. A CRP protein concentration sandwich test method of a digital plasma immunoadsorption kit is characterized by comprising the following steps:
firstly, forming a self-assembled monolayer (self-assembled monolayers) on the surface of a chip in an 11-mercaptodecanoic acid solution, activating carboxyl by EDC and NHS, and binding CRP capture antibody to the surface of the chip; blocking non-specific sites with BSA and ethanolamine solution and covering the remaining NHS; then adding the CRP protein solution to the surface of the chip, so that the CRP protein free in the solution is captured by the CRP antibody fixed on the surface of the chip and remains on the surface of the chip; (ii) allowing the concentration of CRP in the solution to be below a predetermined concentration, wherein on average each nanopore comprises one to a plurality of molecules of CRP protein; the CRP protein is randomly dispersed to all positions on the surface of the chip and captured, so that the CRP protein is distributed on the surface of the chip unevenly; finally, adding a predetermined amount of CRP detection antibody to the surface of the chip to combine with the captured CRP protein, so that the combination of the CRP detection antibody on the surface of the chip changes the RI of the area environment in which the CRP detection antibody is located to red shift the plasma resonance peak, thereby dividing the surface area of the chip into two parts, wherein the area containing the CRP protein changes the original SPR signal after being combined with the CRP detection antibody, and the area without the CRP protein can not combine with the CRP detection antibody to keep the original SPR signal;
the digital plasma immunoadsorption kit comprises: the kit comprises a box body, a nano plasma sensor chip and a reagent, wherein the nano plasma sensor chip and the reagent are arranged in the box body; the surface of the nano plasma sensor chip is provided with a nano cup array, and each nano cup in the nano cup array comprises a nano column, a titanium layer and a gold layer which are sequentially formed on the surface of the nano column;
the preparation method of the nano plasma sensor chip comprises the following steps: manufacturing an original mold by a tapered nano-pillar array manufactured on a silicon oxide wafer through laser interference lithography; silanization of the original mold to obtain hydrophobicity before replication of the original mold; then, coating an optical adhesive on a clean original mold, placing a PET sheet on the top of the mold, and stripping a PET sheet with a nanopore array on the surface from the mold after ultraviolet irradiation; thereafter, a titanium layer and a gold layer were deposited on the PET surface having the nanopore array using an electron beam evaporator to fabricate a nanoplasmon sensor chip.
2. The test method of claim 1, comprising:
introducing a Poisson distribution formula:
Figure FDA0003402664780000011
wherein λ is a parameter associated with the concentration c of the CRP protein, and is k1*c，k1Is a coefficient; the probability P represents the ratio of the nanopore region where the SPR signal changes to the detected nanopore region; the approximation of the ratio of the nanopore region is represented by the change in the number of pixel points, and the rate of change in the number of pixel points where the SPR signal changes is represented as p, i.e., p
Figure FDA0003402664780000021
Ncp represents the number of changed pixels, Ntot represents the total number of pixels; adding a correction factor k2To establish the corresponding relationship between the region where the SPR signal changes and the change of the number of pixel points, there is a formula of the corresponding relationship: p (X ═ 0) ═ 1-k2X p, expressed as a function of p and c, gives:
p＝k2(1-1k1×c)；
taking logarithms at two sides to obtain a concentration formula:
c＝k1×In(1-k2×p)。
3. the test method of claim 2, further comprising: fitting the function relation of the change rate p of the number of the pixel points along with the CRP concentration by using the corresponding relation formula, and determining a coefficient k by taking the fitting result when the CRP concentration is lower than 50ng/ml1、k2To further express the concentration formula as C (CRP) 0.10804 × In (1-4.90938 × p) to calculate the CRP protein concentration.
4. The test method according to claim 1 or 2, wherein the test method is for the detection of the inflammation marker CRP protein.
5. The test method according to claim 1 or 2, wherein the illumination is performed by a halogen lamp, and the image of the transmission mode is exposed on the CCD camera through a condenser lens and a magnifying objective lens.
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